Involvement of the TetR-Type Regulator PaaR in the Regulation ofPristinamycin I Biosynthesis through an Effect on Precursor Supply inStreptomyces pristinaespiralis

Yawei Zhao,a Rongrong Feng,a Guosong Zheng,a Jinzhong Tian,a Lijun Ruan,b Mei Ge,b Weihong Jiang,a,c Yinhua Lua

Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai,People’s Republic of Chinaa; Shanghai Laiyi Center for Biopharmaceuticals R&D, Shanghai, People’s Republic of Chinab; Shanghai Collaborative Innovation Center forBiomanufacturing Technology, Shanghai, People’s Republic of Chinac

ABSTRACT

Pristinamycin I (PI), produced by Streptomyces pristinaespiralis, is a streptogramin type B antibiotic, which contains two pro-teinogenic and five aproteinogenic amino acid precursors. PI is coproduced with pristinamycin II (PII), a member of strepto-gramin type A antibiotics. The PI biosynthetic gene cluster has been cloned and characterized. However, thus far little is under-stood about the regulation of PI biosynthesis. In this study, a TetR family regulator (encoded by SSDG_03033) was identified asplaying a positive role in PI biosynthesis. Its homologue, PaaR, from Corynebacterium glutamicum serves as a transcriptionalrepressor of the paa genes involved in phenylacetic acid (PAA) catabolism. Herein, we also designated the identified regulator asPaaR. Deletion of paaR led to an approximately 70% decrease in PI production but had little effect on PII biosynthesis. Identicalto the function of its homologue from C. glutamicum, PaaR is also involved in the suppression of paa expression. Given thatphenylacetyl coenzyme A (PA-CoA) is the common intermediate of the PAA catabolic pathway and the biosynthetic pathway ofL-phenylglycine (L-Phg), the last amino acid precursor for PI biosynthesis, we proposed that derepression of the transcription ofpaa genes in a �paaR mutant possibly diverts more PA-CoA to the PAA catabolic pathway, thereby with less PA-CoA metabolicflux toward L-Phg formation, thus resulting in lower PI titers. This hypothesis was verified by the observations that PI produc-tion of a �paaR mutant was restored by L-Phg supplementation as well as by deletion of the paaABCDE operon in the �paaRmutant. Altogether, this study provides new insights into the regulation of PI biosynthesis by S. pristinaespiralis.

IMPORTANCE

A better understanding of the regulation mechanisms for antibiotic biosynthesis will provide valuable clues for Streptomycesstrain improvement. Herein, a TetR family regulator PaaR, which serves as the repressor of the transcription of paa genes in-volved in phenylacetic acid (PAA) catabolism, was identified as playing a positive role in the regulation of pristinamycin I (PI) byaffecting the supply of one of seven amino acid precursors, L-phenylglycine, in Streptomyces pristinaespiralis. To our knowledge,this is the first report describing the interplay between PAA catabolism and antibiotic biosynthesis in Streptomyces strains. Con-sidering that the PAA catabolic pathway and its regulation by PaaR are widespread in antibiotic-producing actinomycetes, itcould be suggested that PaaR-dependent regulation of antibiotic biosynthesis might commonly exist.

Pristinamycin I (PI), produced by Streptomyces pristinaespira-lis, is a branched cyclohexadepsipeptide antibiotic and be-

longs to the B group of streptogramins. PI is coproduced withpristinamycin II (PII), a polyunsaturated cyclopeptidic macrolac-tone antibiotic, which is a member of the A group of strepto-gramin antibiotics (1, 2). Two substances are produced in a 30:70ratio and show a potent synergistic effect with an approximately100-fold-higher bactericidal activity than that from treatmentwith a single component alone (3). The combined application ofPI and PII semisynthetic derivatives, quinupristin-dalfopristin, isvery active against many Gram-positive bacteria, including mul-tidrug-resistant pathogens, such as methicillin-resistant Staphylo-coccus aureus (MRSA), drug-resistant Streptococcus pneumoniae,and vancomycin-resistant Enterococcus faecium (4).

PI biosynthesis is catalyzed by a nonribosomal peptide synthetase(NRPS) complex composed of SnaA, SnaC, and SnaDE, which areresponsible for the successive condensation of two proteinogenicamino acids, L-threonine and L-proline, and five aproteinogenicamino acids, including 3-hydroxypicolinic acid, L-aminobutyric acid,4-oxo-L-pipecolic acid, L-phenylglycine (L-Phg), and 4-N,N-dimeth-ylaminoL-phenylalanine (DMAPA) or N-methyl-4-(methylamino)-

L-phenylalanine (MMAPA) (5). In the PI biosynthetic gene cluster,12 genes are involved in PI precursor supply, including hpaA, pipAand snbF and gene clusters papACBM and pglABCDE, which are re-quired for the formation of 3-hydroxypicolinic acid, 4-oxo-L-pipe-colic, DMAPA (or MMAPA), and L-Phg, respectively (5, 6). Al-though a possible biosynthetic pathway has been proposed, little is

Received 21 January 2015 Accepted 30 March 2015

Accepted manuscript posted online 13 April 2015

Citation Zhao Y, Feng R, Zheng G, Tian J, Ruan L, Ge M, Jiang W, Lu Y. 2015.Involvement of the TetR-type regulator PaaR in the regulation of pristinamycin Ibiosynthesis through an effect on precursor supply in Streptomycespristinaespiralis. J Bacteriol 197:2062–2071. doi:10.1128/JB.00045-15.

Editor: T. M. Henkin

Address correspondence to Yinhua Lu, yhlu@sibs.ac.cn.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00045-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00045-15

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understood about the regulation of PI biosynthesis in S. pristinaespi-ralis. To our knowledge, until now, only seven cluster-situatedregulatory genes (from papR1 to papR6 and spbR) and a serine/threonine protein kinase gene, spy1, were identified as being in-volved in the regulation of PI biosynthesis (2, 5, 7–9). For thegenus Streptomyces, manipulating the regulatory network thatcontrols antibiotic biosynthesis has been proven to be an efficientmethod for improving the production levels of desired antibiotics(10). Therefore, a better understanding of the regulation mecha-nisms for PI biosynthesis will provide valuable clues for strainimprovement to increase PI titer by metabolic engineering ap-proaches.

In our previous work, we generated a dozen S. pristinaespiralismutants with the individual deletion of a number of TetR familyregulatory genes and have checked their effects on pristinamycinproduction (unpublished data). A mutant with significantly re-duced PI production was that with the deletion of SSDG_03033.The regulator encoded by SSDG_03033 is highly conserved in Ac-tinobacteria and shows a high amino acid sequence identity withPaaR from Corynebacterium glutamicum, which functions as a re-pressor of the phenylacetic acid (PAA) degradation pathway (11).Thus, we also named the SSDG_03033-encoding regulator PaaR.Herein, the mechanism underlying its function in PI productionwas revealed. We demonstrated that PaaR plays a positive role in

PI biosynthesis by repressing the transcription of paa genes in-volved in PAA catabolism and thereby possibly diverting phenyl-acetyl coenzyme A (PA-CoA) to the biosynthetic pathway of L-Phg, the last building block for PI biosynthesis. PA-CoA is thecommon intermediate for both the PAA degradation pathway andthe L-Phg biosynthetic pathway, as shown in Fig. 1 (6, 11).

MATERIALS AND METHODSBacterial strains, growth conditions, plasmids. The bacterial strains andplasmids used in this study are listed in Table 1. Escherichia coli strains,including DH5�, BL21(DE3), and ET12567/pUZ8002, were cultivated inLuria-Bertani (LB) medium at 37°C. DH5� was used as the host for rou-tine molecular cloning. ET12567/pUZ8002 was used as the donor strain inthe intergeneric conjugation. BL21(DE3) was employed for protein over-expression. Antibiotics, including ampicillin (100 �g/ml), apramycin (50�g/ml), kanamycin (50 �g/ml), and/or chloramphenicol (25 �g/ml),were added to the medium when appropriate.

S. pristinaespiralis HCCB10218 (CGMCC 5486), which was isolatedafter physical and chemical treatments of ATCC 25486, was used as theoriginal strain. S. pristinaespiralis strains were grown at 30°C in RP liquidmedium (peptone, 5; yeast extract, 5; valine, 0.5; NaCl, 2; KH2PO4, 0.5;MgSO4·7H2O, 1[in grams per liter]; pH 6.4) for genomic DNA isolation(8). RP agar medium was used for the preparation of spore suspensionsand conjugal transfer between E. coli and S. pristinaespiralis (12). For the S.pristinaespiralis fermentation, seed medium and fermentation medium

FIG 1 Schematic presentation of the proposed phenylacetic acid (PAA) degradation pathway and L-Phg biosynthetic pathway. The PAA degradation pathwayand L-Phg biosynthetic pathway are indicated by blue and red arrows, respectively. PglA, hydroxyacyl-dehydrogenase; PglB, pyruvate dehydrogenase E1component �-subunit; PglC, pyruvate dehydrogenase E1 component �-subunit; PglD, thioesterase type II; PglE, phenylglycine aminotransferase; SnbE, pristi-namycin I (PI) synthase 3 and 4; PaaABCDE, ring 1,2-phenylacetyl-CoA epoxidase subunits; PaaF, 2,3-dehydroadipyl-CoA hydratase; PaaG, ring 1,2-epoxy-phenylacetyl-CoA isomerase, oxepin-CoA forming; PaaH, 3-hydroxyadipyl-CoA dehydrogenase; PaaJ, 3-oxoadipyl-CoA/3-oxo-5,6-dehydrosuberyl-CoA thio-lase; PaaK, phenylacetyl-CoA ligase; PaaZ, fused oxepin-CoA hydrolase–3-oxo-5,6-dehydrosuberyl-CoA semialdehyde dehydrogenase.

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were prepared as described previously (5). When necessary, apramycin(50 �g/ml), kanamycin (50 �g/ml), and/or thiostrepton (50 �g/ml) wasadded.

Construction of the �paaR and �paaR �paaABCDE deletion mu-tants. A �paaR mutant with an in-frame deletion of the DNA sequenceencoding amino acids ranging from position 38 to position 195 of thePaaR regulator was constructed on the basis of the parental strain S. pris-tinaespiralis HCCB10218 by traditional homologous recombination (12).The upstream and downstream regions (1,243 and 1,249 bp, respectively)of the target DNA sequence were amplified from the genomic DNA ofHCCB10218 by using primer pairs paaRup-fw/rv and paaRdown-fw/rv(see Table S1 in the supplemental material). The two PCR products weredigested with HindIII/XbaI and XbaI/EcoRV, respectively, and clonedinto the replication temperature-sensitive plasmid pKC1139 betweenHindIII and EcoRV to yield pKC-paaR. The construct was introducedinto ET12567/pUZ8002 and subsequently transferred to the parentalstrain HCCB10218 by conjugal transfer. To obtain the single-cross-over strains, apramycin-resistant strains were passaged three times onRP agar plates containing apramycin at 37°C. The single crossoverswere checked by PCR using the primer pair pKC1139-F/paaRSC-rv(see Table S1). The above obtained strains were inoculated into apra-mycin-free RP liquid medium and passaged for three rounds. Subse-quently, the cultures were serially diluted and plated on apramycin-

free RP agar medium. To identify the double-crossover strains, thesingle colonies were picked and grown on apramycin-free and -sup-plemented RP agar plates by replica plating. The �paaR mutant strainswere checked by PCR using the primer pair paaRSJ-fw/rv (see TableS1), followed by DNA sequencing.

A �paaR �paaABCDE double mutant with the deletion of the en-tire paaABCDE gene cluster based on the �paaR mutant was con-structed by an I-SceI-mediated homologous recombination (13). Theupstream and downstream homologous arms (1,757 and 1,937 bp,respectively) were amplified with primer pairs paaA-Eup-fw/rv andpaaA-Edown-fw/rv (see Table S1 in the supplemental material). ThePCR products were treated with SwaI/XbaI and XbaI/PacI, respec-tively, and ligated to plasmid pKC1139-SPI between SwaI and PacI toyield pKC-paaABCDE (Table 1). The construct was introduced intothe �paaR mutant by conjugal transfer. Apramycin-resistant strainswere passaged three times on RP agar supplemented with apramycin.The single-crossover events were confirmed by PCR using the primerpair pKC1139-F and paaA-ESC-rv (see Table S1). After isolation of thespores of the single-crossover strains, the plasmid pALSceI (Table 1),which contains the codon-optimized I-SceI-encoding gene under thecontrol of an inducible promoter tipAp, was introduced by conjugaltransfer and I-SceI expression was induced by thiostrepton. Thiostrep-ton-resistant strains were checked for double crossovers by replica

TABLE 1 Strains and plasmids used in this study

Strain or plasmid Genotype or description Reference

StrainsS. pristinaespiralis

HCCB10218 Parental strain Provided by ShanghaiLaiyi Center

�paaR mutant Mutant with in-frame deletion of the paaR gene in the genome of HCCB10218 This study�paaR �paaABCDE mutant Double mutant with in-frame deletion of the gene cluster paaABCDE based on the �paaR

mutantThis study

10218/pIB139 HCCB10218 with the empty vector pIB139 This study�paaR/pIB139 mutant �paaR mutant with the empty vector pIB139 This study�paaR/pIB-paaR mutant �paaR mutant with the complemented vector pIB-paaR This study

E. coliDH5a F� �80lacZ�M15 �(lacZYA-argF) U169 deoR recA1 endA1 hsdR17 supE44 �� thi-1 gyrA96

relA1GIBCO-BRL

ET12567/pUZ8002 dam-13::Tn9 dcm-6 hsdM; containing the nontransmissible RP4 derivative plasmid pUZ8002 20BL21(DE3) F� ompT hsdS gal dcm Novagen

PlasmidspMD18-T simple TA cloning vector TaKaRapIB139 Expression vector modified from the integrative vector pSET152, harboring a strong

constitutive promoter, ermE*p21

pIB-paaR Recombinant plasmid harboring the paaR gene sequence cloned in the pIB139 plasmid betweenthe NdeI and EcoRI sites

This study

pKC1139 Replicative vector for actinomycetes containing the temperature-sensitive replicon pSG5; oriTaac(3)IV

22

pKC1139-SPI Derivative of pKC1139 by adding two enzyme sites, SwaI and PacI, and the I-SceI recognitionsequence

This study

pKC-paaR Recombinant vector for constructing the �paaR mutant with two homologous arms (1,180 bpand 1,244 bp), which were cloned in the temperature-sensitive plasmid pKC1139 betweenthe HindIII and EcoRV sites

This study

pKC-paaABCDE Recombinant vector for the deletion of paaABCDE based on the �paaR mutant, carrying twohomologous arms (1,757 bp and 1,937 bp), which were cloned in the temperature-sensitiveplasmid pKC1139-SPI between the SwaI and PacI sites

This study

pALSceI E. coli-Streptomyces shuttle plasmid containing the I-SceI-encoding gene under the control ofthe tipA promoter, oriT Hygr

This study

pET28a Expression vector NovagenpET-paaR Recombinant plasmid with the cloning of the paaR gene sequence in pET28a between the NdeI

and EcoRI sitesThis study

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plating. The mutant strains were verified by PCR using the primerspaaA-ESJP-fw/rv and paaA-E-SJN-fw/rv (see Table S1). The resultingstrains were passaged three times on RP agar medium without antibi-otics to remove plasmid pALSceI, resulting in the �paaR �paaABCDEmutant.

Complementation assay. The paaR opening reading frame (ORF; 624bp) was amplified from the genomic DNA of HCCB10218 using theprimer pair paaRex-fw/rv (see Table S1 in the supplemental material).After treatment with NdeI and EcoRI, the resultant PCR product wasligated to plasmid pIB139, generating pIB-paaR, in which paaR expres-sion was under the control of a strong constitutive promoter ermE*p. Theobtained plasmid was transferred to the �paaR mutant by conjugal trans-fer, resulting in the �paaR/pIB-paaR complemented strain. Two otherstrains, 10218/pIB139 and the �paaR/pIB139 mutant, were constructedas controls by introducing the empty plasmid pIB139 into HCCB10218and the �paaR mutant, respectively.

S. pristinaespiralis fermentation and analysis of pristinamycin pro-duction. S. pristinaespiralis fermentation and analysis of pristinamycinproduction were carried out according to the previously described meth-ods with some modifications (5, 8). Briefly, the S. pristinaespiralis strainswere grown on RP agar medium at 30°C for 4 to 5 days. Subsequently, theagar cultures were inoculated into 25 ml of seed medium in 250-ml flasksat 27°C and 240 rpm. After incubation for 40 to 44 h, 2 ml of the seedcultures was transferred into 25 ml of fermentation medium in 250-mlflasks. Fermentation samples (0.5 ml each) were collected at 48 and 72 h,respectively, and extracted with the same volume of acetone for 60 min.The mixtures were centrifuged at 12,000 rpm for 5 min, and pristinamycinproduction in the supernatants was analyzed by bioassay and high-per-formance liquid chromatography (HPLC). In the bioassay, Bacillus subti-lis ATCC 6633 was used as the indicator strain. HPLC analysis of pristi-namycin production was performed as described previously (8). Thestandard curves for pristinamycin quantitative analysis were made usingpurified PIa and PIIa (98%) obtained from Laiyi Co. Ltd. (Shanghai,China).

RNA isolation and qPCR analysis. Fermentation samples of S. pristi-naespiralis were collected at 48 h. After being frozen in liquid nitrogen, thecells were ground into powder. RNA samples were prepared using anultrapure RNA kit (Cwbio, Shanghai, China). RNase-free DNase I(TaKaRa, Dalian, China) was utilized to remove the residual genomicDNA. The integrity and quality of RNA samples were analyzed by 1.5%agarose gel electrophoresis, and the quantity was measured with a Nano-Drop 2000 spectrophotometer (Thermo Scientific, USA). RNA reversetranscription was performed using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, USA). Quantitative real-time re-verse transcription (RT)-PCR (qPCR) analysis was performed as de-scribed previously (8). The primer pairs used for qPCR are listed in TableS2 in the supplemental material. The reactions were performed in aMyiQ2 PCR machine (Bio-Rad, USA) with the following parameters:DNA denaturation at 95°C for 2 min and 40 cycles of 95°C for 20 s, 60°Cfor 20 s, and 72°C for 20 s. PCRs were conducted in triplicate for eachgene. The relative expression levels of the tested genes were normalized tohrdB (SSDG_06142, encoding a housekeeping sigma factor) and were de-termined using the 2���CT method (14). The relative value for the expres-sion of each gene in the parental strain HCCB10218 was assigned as 1.qPCR analysis was performed with three independent RNA samples (bi-ological replicates), and error bars represent the standard deviations (SD).

Determination of the TSSs. 5=-RACE (rapid isolation of cDNA end)experiments were performed using the 5=-Full RACE kit with TAP(TaKaRa, Dalian, China) according to the instructions provided by themanufacturer. Apart from the primers packaged in the kit, the gene-spe-cific primers are listed in Table S2 in the supplemental material. After tworounds of PCR amplification (including a nested PCR), the 5=-RACEproducts were cloned into the pMD-18T vector (TaKaRa, Dalian, China).Ten plasmids isolated from the transformants were sequenced, and the

specific transcriptional start sites (TSSs) were determined if more thanseven of the sequenced cDNAs had identical 5= ends.

Overexpression and purification of the recombinant PaaR protein.The entire paaR gene sequence was amplified from HCCB10218 genomicDNA using the primers paaRex-fw/rv (see Table S1 in the supplementalmaterial). After treatment with NdeI and EcoRI, the PCR product wasligated to the expression vector pET28a, resulting in pET-paaR. The cor-rect recombinant plasmids were verified by DNA sequencing. The ob-tained plasmid pET-paaR was transformed into E. coli BL21(DE3) com-petent cells. The overexpression of His6-PaaR was induced by addingIPTG (isopropyl-�-D-thiogalactopyranoside) to a concentration of 500�M, and the cultures were grown at 16°C overnight. Cells were collectedby centrifugation at 5,000 rpm for 10 min and resuspended in the bindingbuffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM imidazole, 10%glycerol). The cell suspension was disrupted using a French press (Con-stant Systems Limited, United Kingdom), and cell debris was removed bycentrifugation. His6-PaaR was purified using a Ni-nitrilotriacetic acid(Ni-NTA)–agarose column (GE Health Care, Sweden) and eluted withincreasing concentrations of imidazole from 10 mM to 500 mM. Theconcentration of His6-PaaR was determined using a Bradford kit (Sangon,China), and His6-PaaR purity was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE).

EMSA. Electrophoretic mobility shift assays (EMSAs) between puri-fied His6-PaaR and the tested probes (see Table S3 in the supplementalmaterial) were carried out using a previously described method (15).Briefly, DNA fragments containing the respective promoter regions wereamplified from HCCB10218 genomic DNA using the primer pairs listedin Table S1 in the supplemental material. Cy5-labeled oligonucleotide(5=-AGCCAGTGGCGATAAG-3=) was used in the second-round PCR tomake the labeled probes. The labeled probes (10 ng) were incubated withdifferent amounts of purified His6-PaaR at 25°C for 20 min in the bindingbuffer as described before (8). The intergenic region between SSDG_03653 and SSDG_05964 that contains no putative PaaR-binding motifwas tested as a negative control. The specificity of the DNA-protein inter-actions was verified by adding 2 �g of unlabeled probes or salmon spermDNA. The unlabeled DNAs were mixed with His6-PaaR at 25°C for 20 minbefore the labeled probes were added. Finally, the reaction mixtures wereseparated on 1.5% Tris-acetate-EDTA (TAE)–agarose gels in 0.5 TAEbuffer. After 1 h of electrophoresis at 4°C, the gels were scanned using aFLA-9000 phosphorimager (Fujifilm, Japan).

RESULTSBioinformatical analysis of PaaR, a TetR family regulator en-coded by SSDG_03033. The SSDG_03033 gene encodes a TetRfamily regulator, and its homologues are widespread in Actino-bacteria. BLAST analysis revealed that this regulator exhibits highamino acid sequence identities with its homologues from otherStreptomyces strains (78 to 89%; the maximum number ofaligned sequences to display is set at 100) (data not shown). Weshow the alignment of the deduced amino acid sequences ofthe SSDG_03033-encoded regulator and its seven homologuesfrom the Streptomyces whole genomes available online (http://streptomyces.org.uk/) (see Fig. S1 in the supplemental mate-rial). These eight regulators harbor nearly identical TetR N do-mains, which are involved in DNA binding. The functions of thesehomologues are yet uncharacterized. In actinomycetes, thus far,only PaaR from C. glutamicum, a homologue of the SSDG_03033-encoded regulator, has been functionally identified, which is in-volved in suppression of the transcription of the paa genes encod-ing the phenylacetic acid (PAA) catabolism pathway (11). Herein,we also name the SSDG_03033-encoded regulator PaaR.

Similar to that in C. glutamicum, the paaR gene in S. pristi-naespiralis is clustered with the paa genes (Fig. 2). However, the

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organization of the paa gene cluster in S. pristinaespiralis is appar-ently different from that in C. glutamicum (11), although a coher-ent paaABCDE operon was found in the genome of S. pristinaespi-ralis. We also found that in comparison with C. glutamicum, allpaa genes are clustered together in S. pristinaespiralis, some paagenes, such as paaI and paaK, are scattered elsewhere, and nopaaFGJYT homologous genes were found. Interestingly, paaR inS. pristinaespiralis is located immediately adjacent to bkdR and thebkdABC operon, which encode a leucine response regulator and abranched-chain amino acid dehydrogenase complex, respectively(16). Further bioinformatical analysis revealed that the geneticorganization of paa genes may be relatively conserved in differentStreptomyces strains (see Fig. S2 in the supplemental material). It isworth noting that a highly conserved bkdABC-bkdR-paaR gene

cluster was observed in the genomes of all selected Streptomycesstrains, suggesting that PaaR is likely to participate in the regula-tion of branched-chain amino acid catabolism in Streptomyces.

Deletion of paaR affects only PI biosynthesis. We con-structed a �paaR mutant with partial deletion of the paaR genesequence based on the parental strain HCCB10218, and pristina-mycin production was assayed by both a bioassay and HPLC anal-ysis. The fermentation samples were collected at 48 and 72 h. Littledifference in bacterial growth was observed between the parentalstrain and the �paaR mutant (data not shown). The bioassay (us-ing the supernatants extracted from fermentation samples col-lected at 48 h and B. subtilis as the indicator strain) showed that theinhibition zone produced by the �paaR mutant is much smallerthan that of the parental strain (Fig. 3A). Further quantitative

FIG 2 Genetic organization of the paa gene cluster in the genomes of S. pristinaespiralis and C. glutamicum. Homologous paa genes in these two strains areindicated in the same color. The bkdR and bkdABC genes are represented by green and yellow, respectively. The genes that have no relationship with the PAAdegradation pathway are represented by gray. The probes containing the corresponding intergenic regions bound by purified PaaR protein in the EMSA analysisare indicated by vertical red arrows, and the probe with no interaction with PaaR is indicated by a vertical gray arrow. The PaaR-binding sites are represented byred dots. bkdR encodes a leucine-response regulator; bkdABC encode a branched-chain amino acid dehydrogenase complex; paaT, paaI, and paaY encode aputative transporter, a thioesterase, and an acetyltransferase, respectively. The functions of other paa genes are as described in the legend for Fig. 1.

FIG 3 Effects of paaR deletion on PI production. (A) Bioassay of pristinamycin production using B. subtilis as the indicator strain. Fermentation samples werecollected at 48 h. (B) HPLC analysis of pristinamycin production. PIa and PIIa are the main components of PI and PII, respectively. Fermentation samples werecollected at two time points (48 and 72 h). Error bars represent the standard deviations from three biological replicates. Five strains, including the parental strainHCCB10218, the �paaR mutant, HCCB10218 containing the control vector (10218/pIB139), the �paaR mutant containing the control vector (�paaR/pIB139),and the complemented strain (�paaR/pIB-paaR), were tested.

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analysis of pristinamycin production by HPLC revealed that dele-tion of paaR resulted in a 3.3-fold reduction in PIa (the majorcomponent of PI) production. However, little effect on PIIa (themajor component of PII) production was detected upon paaRinactivation (Fig. 3B). Introduction of the wild-type paaR gene(under the control of a strong constitutive promoter ermE*p on anintegrative plasmid pIB139) into the �paaR mutant could easilyrestore PIa production and the inhibition zone to the levels of theparental strain HCCB10218 (with the control vector pIB139) (Fig.3A and B). These results clearly demonstrate that PaaR is involvedin activation of PI biosynthesis in S. pristinaespiralis.

PaaR functions positively in PI production by affecting thetranscription of the paa genes involved in PAA degradation.Given that the PaaR homologue in C. glutamicum functions as arepressor of the transcription of the paa genes and that, moreover,the PaaR-binding motif was detected in the promoter regions ofthe paa genes in three Streptomyces strains, including Streptomycescoelicolor, Streptomyces avermitilis, and Streptomyces griseus (11),we speculated that PaaR in S. pristinaespiralis may be also impli-cated in the suppression of paa gene transcription. As shown inFig. 1, phenylacetyl-CoA (PA-CoA) is the common intermediateof the L-Phg biosynthetic pathway and PAA catabolic pathway(Fig. 1). If PaaR exerts a repressor role in paa expression as pro-posed above, its inactivation would enhance the transcription ofthese paa genes and thereby result in more consumption of PA-CoA by the PAA degradation pathway and accordingly reduce theformation of L-Phg as well as PI.

To explore this possibility, as the first step, we compared thetranscription of the paa genes in the �paaR mutant with that inHCCB10218 using qPCR analysis. Five paa genes, including paaH,paaZ, paaA, paaK, and paaI, were selected and tested. Four genes,including two PI biosynthetic genes (hpaA and pglC), one PIIbiosynthetic gene, snaN, and paaR itself, were tested as negativecontrols. In addition, as paaR is clustered with bkdR and bkdABC,the transcription of bkdR and bkdA was also analyzed to assesswhether PaaR is involved in the regulation of these bkd genes.RNA samples were isolated from fermentation samples collectedat 48 h. Transcriptional analysis revealed that the mRNA abun-dance of four paa genes (paaH, paaZ, paaA, and paaK) was en-hanced at least 10-fold after paaR deletion. Deletion of paaR alsoresulted in enhanced expression of bkdA and bkdR by 2- and4-fold, respectively. However, little difference was observed in thetranscription of paaI and four PI/PII biosynthetic genes in the�paaR mutant compared with that in the parental strain (Fig. 4).Moreover, as expected, no paaR transcription was detected in the�paaR mutant. These data clearly demonstrated that PaaR re-pressed the transcription of the majority of paa genes as well asbkdA and bkdR in S. pristinaespiralis and suggested that the role ofPaaR in PI biosynthesis is independent of the PI biosyntheticgenes. Since paaH overlaps with paaR by 33 bp (Fig. 2), it is likelythat PaaR regulates its own transcription from the promoter up-stream of paaH. In addition, considering the functions of bkdABCand bkdR in S. coelicolor as described previously (16), we couldconclude that PaaR is also involved in branched-chain amino acidmetabolism. It should be noted that the degree of transcriptionalupregulation of bkdA is lower than that of bkdR upon paaR dele-tion (Fig. 4). As described before, bkdR exerted a negative effect onbkdABC transcription (16); thus, it could be proposed that therelatively lower transcriptional upregulation of bkdA in the �paaRmutant may be the result of the combined role of PaaR and BkdR.

Subsequently, we deleted the paaABCDE operon based on the�paaR mutant, generating a �paaR �paaABCDE double mutant,to verify that the reduced PI production in the �paaR mutant isdue to derepression of paa transcription. Pristinamycin produc-tion was measured by bioassay and HPLC analysis. Three strains,including the parental strain HCCB10218, the �paaR mutant, andthe �paaR �paaABCDE double mutant, were grown in fermenta-tion medium, and samples were collected at 48 and 72 h. As ex-pected, the results from the bioassay (fermentation samples col-lected at 48 h) revealed that unlike the �paaR mutant, whichproduced a small inhibition zone, the �paaR �paaABCDE doublemutant and the parental strain produced comparable clear zones(Fig. 5A). Further HPLC analysis showed that in comparison withthe �paaR mutant, which produced only PI levels that were about30% of those of the parental strain, the �paaR �paaABCDE dou-ble mutant produced nearly the same amounts of PIa as those ofthe parental strain at the tested time points (Fig. 5B). Interestingly,compared with both the parental strain and the �paaR mutant,the �paaR �paaABCDE double mutant produced 20% lower PIIalevels. Nevertheless, these results demonstrated that reduced PIproduction in the �paaR mutant is due to the enhanced expres-sion of paa genes encoding the PAA degradation pathway.

Addition of L-Phg to the �paaR mutant partially restores PIformation. To confirm that the reduced PI formation is due to thedecreased L-Phg supply, we performed supplementation experi-ments by adding L-Phg to the fermentation medium of the �paaRmutant and the parental strain HCCB10218. These two strainswere incubated in seed medium for 44 to 48 h and were subse-quently transferred into 25 ml of fermentation medium supple-mented with 10 mg L-Phg (Sigma-Aldrich, MO, USA). The sametwo strains without the addition of L-Phg were tested as negativecontrols. After incubation in fermentation medium for 48 and 72

FIG 4 Effects of paaR deletion on the transcription of the paa genes. RNAsamples were prepared from the fermentation cultures of the parental strainHCCB10218 and the �paaR mutant (collected at 48 h). The transcriptionlevels for each tested gene were normalized to the internal control, the hrdBgene. The values for the transcription of each gene in the parental strain werearbitrarily assigned as 1. The relative transcription levels are averages of threeindependent biological replicates, and error bars represent the standard devi-ations.

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h, fermentation samples (0.5 ml) were collected and pristinamy-cin production was analyzed by both bioassay and HPLC. Bioassayshowed that the addition of L-Phg to the �paaR mutant resulted ina much larger clear zone than that of the mutant without theaddition of L-Phg (samples collected at 48 h). For the parentalstrain, intriguingly, we observed that addition of L-Phg resulted ina slightly smaller inhibition zone than that without L-Phg (Fig.6A). Further HPLC analysis revealed that, as shown in Fig. 6B,L-Phg addition to the �paaR mutant could partially restore PIproduction at the tested time points; PI production was increasedabout 2-fold (from 15 to 30 mg/liter). However, to our surprise,L-Phg addition to the mutant resulted in enhanced PII productionby around 20%. For the parental strain, addition of L-Phg led to anintriguingly enhanced PII but reduced PI production at the testedtime points (Fig. 6B). Since the synergistic bacteriostatic activity ofPI and PII is possibly dependent on the ratios of these two com-

ponents (17), we speculated that a higher ratio of PII to PI mayexplain why the parental strain supplemented with L-Phg (PII:PIratio � 10:1) produced a slightly smaller clear zone than thatwithout L-Phg (PII:PI ratio � 4:1) (Fig. 6A).

PaaR represses the transcription of paa genes directly. A pre-vious report has revealed that PaaR from Actinobacteria recog-nizes a conserved, perfect palindromic motif (5=-ACCGA-n4-TCGGT-3=); moreover, in three Streptomyces strains, including S.coelicolor, S. avermitilis, and S. griseus, the identified PaaR-bindingmotifs were predicted to be located in the promoter regions of thepaa genes as well as in the intergenic region of bkdA-bkdR (11). Inthis study, to determine whether it is the same case in S. pristi-naespiralis, we searched for this signature sequence in the inter-genic regions of paaH-paaZ, paaA-SSDG_03038, paaK-SSDG_07468, and bkdA-bkdR, which harbors the promoter regions ofpaaH and paaZ, paaA, paaK, and bkdA and bkdR, respectively. In

FIG 5 Effects of deletion of both paaR and paaABCDE on pristinamycin production. (A) Bioassay of pristinamycin production using B. subtilis as the indicatorstrain. Fermentation samples were collected at 48 h. (B) HPLC analysis of pristinamycin production. PIa and PIIa are the main components of PI and PII,respectively. Fermentation samples were collected at two time points (48 and 72 h). Error bars represent the standard deviations from three biological replicates.Three strains, including the parental strain HCCB10218, the �paaR mutant, and the �paaR �paaABCDE double mutant (�paaR/ABCDE), were tested.

FIG 6 Effects of L-Phg addition on pristinamycin production of the �paaR mutant. (A) Bioassay of pristinamycin production using B. subtilis as the indicatorstrain. Fermentation samples with or without L-Phg supplementation were collected at 48 h. (B) HPLC analysis of pristinamycin production. PIa and PIIa are themain components of PI and PII, respectively. Fermentation samples were harvested at two time points (48 and 72 h). Error bars represent the standard deviationsfrom three biological replicates. Two strains (the parental strain HCCB10218 and the �paaR mutant) were tested under the condition of fermentation mediumwith or without the addition of L-Phg (10 mg/25 ml medium).

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addition, according to the genetic organization of paaI and itsneighboring genes, we speculated that paaI is cotranscribed withthe upstream four genes (from SSDG_03653 to SSDG_3650).Thus, the intergenic region of SSDG_03653-SSDG_05964 was alsoexamined. The analysis revealed that the intergenic regions ofpaaH-paaZ, paaA-SSDG_03038, paaK-SSDG_07468, and bkdA-bkdR have the same conserved, perfect palindromic motif (5=-ACCGA-n4-TCGGT-3=) as that identified in C. glutamicum (11). In-terestingly, in the former two regions (paaH-paaZ, paaA-SSDG_03038), we also found another imperfect palindromicmotif (5=-AACGA-n4-TCGGT-3=), which overlaps with the per-fect motif (Fig. 7A). No such motifs were detected in the intergenicregion of SSDG_03653-SSDG_05964, which may explain whypaaR deletion has no effect on paaI transcription. By using com-parative genomic analysis, two such conserved PaaR-binding mo-tifs were detected in the intergenic regions of the paa genes andbetween bdkA and bkdR in the genomes of six other Streptomycesstrains, whose genome sequences are available online (http://streptomyces.org.uk/) (see Table S4 in the supplemental mate-rial), further confirming that PaaR-mediated control of thesegenes is common in Streptomyces strains.

Subsequently, EMSA analysis was performed with purifiedPaaR protein to investigate whether PaaR could specifically bindto these intergenic regions containing the signature sequences. Intotal, four probes containing the intergenic regions of paaH-paaZ,paaA-SSDG_03038, paaK-SSDG_07468, and bkdA-bkdR, respec-tively, were checked in the EMSA analysis(see Table S3 in thesupplemental material). The intergenic region of SSDG_03653-SSDG_05964 that contains no putative PaaR-binding motif wastested as a negative control. As expected, purified PaaR proteincould interact specifically with four tested probes (Fig. 7B) but notwith the negative control, suggesting that the transcription ofthese paa genes was directly regulated by PaaR. To test whetherPaaR was also involved in regulation of the transcription of twofunctionally unidentified genes, SSDG_03038 and SSDG_07468,which are transcribed divergently from paaA and paaK, respec-tively, qPCR was performed and revealed that PaaR repressed thetranscription of SSDG_03038 as well and has no effect on SSDG_07468 expression (Fig. 4).

Furthermore, to determine whether as in C. glutamicum, thePaaR effector ligand is PA-CoA (11), we performed EMSA analy-sis of PaaR and the probe paaA-SSDG_03038p in the presence ofPA-CoA (at concentrations of 0, 10, 25, 50, 100, 200, and 400�M). PAA and acetyl-CoA at the same concentrations were testedas negative controls. As shown in Fig. 7C, we observed that theaddition of increasing concentrations of PA-CoA could eventuallyabolish the binding activity of PaaR for paaA-SSDG_03038p; how-ever, no effects were detected after adding either PAA or acetyl-CoA under the same EMSA conditions. These results confirmedthat PA-CoA acts as the PaaR effector ligand.

Finally, to determine the positions of the PaaR-binding siteswith respect to paa gene promoters, we performed 5=-RACE anal-ysis to identify the putative transcriptional start sites (TSSs) ofthese paa genes that are directly regulated by PaaR, includingpaaH, paaZ, paaA, and paaK. RNA samples were isolated fromfermentation cultures of the �paaR mutant collected at 30 h. Wesuccessfully determined the TSSs of three paa genes (paaA, paaK,and paaZ), and the putative promoter structures were predicted,as shown in Fig. 7A and in Fig. S3 in the supplemental material.Additionally, according to the identified TSS of bkdA2 in S. coeli-

color (a homologue of bkdA in S. pristinaespiralis) (16), we foundthe TSS and �10 and �35 regions of the bkdA promoter. Based onthese results, we observed that the PaaR-binding sites are locatedeither downstream of �10 regions or between �35 and �10 re-gions of the promoters of its target genes, which would hinder thebinding of RNA polymerase to the promoter regions and therebyinhibit gene transcription.

DISCUSSION

In this study, a TetR family regulator, PaaR, was identified as beinginvolved in the regulation of pristinamycin I (PI) by affecting thesupply of one of seven amino acid precursors, L-Phg, in S. pristi-naespiralis. A possible model for PaaR-mediated regulation of PIproduction was proposed as follows. Briefly, PaaR serves as a re-pressor in the expression of paa genes encoding the PAA degrada-tion pathway. Its inactivation results in enhanced paa transcrip-tion, which would result in more consumption of PA-CoA, thecommon intermediate for the L-Phg biosynthetic pathway andPAA catabolic pathway (6, 11) (Fig. 1). Therefore, in the �paaRmutant, the metabolic flux of PA-CoA into the L-Phg biosyntheticpathway was reduced, leading to a lower level of L-Phg and, ac-cordingly, reduced PI biosynthesis. To our knowledge, this is thefirst report describing the interplay between the PAA degradationpathway and antibiotic biosynthesis in Streptomyces strains. Aspreviously reported, the PAA catabolic pathway and its regulationby PaaR are widespread in antibiotic-producing actinomycetes(11). In addition, in bacteria, a substantial part of aromatic com-pounds (such as L-phenylalanine) is catabolized via the PAA path-way (18). Therefore, we speculated that PaaR-dependent regula-tion of antibiotic biosynthesis (particularly for antibiotics usingaromatic compounds as the building precursors) may commonlyexist.

Interestingly, although inactivation of paaABCDE based on the�paaR mutant could restore PI titers comparable to those of theparental strain, the resulting �paaR �paaABCDE double mutantproduced approximately 20% lower PIIa titers than the parentalstrain (Fig. 5). Given that a substantial part of aromatic com-pounds is degraded by the PAA catabolic pathway to acetyl-CoA(18), deletion of paaABCDE may lead to reduced acetyl-CoA for-mation and accordingly a lower level of malonyl-CoA, whichserves as one of the extender units for PII biosynthesis (5). Thedecreased malonyl-CoA formation may account for the reducedPII biosynthesis upon paaABCDE deletion. Now, another ques-tion arises: why did the �paaR mutant, with massively enhancedexpression of the paa genes, still produce the same PII levels asthose of the parental strain? We speculated that the expression ofpaa genes in the parental strain is sufficient for the degradation ofaromatic compounds. Therefore, enhanced paa gene expressionin the �paaR mutant would not result in a significantly increasein malonyl-CoA level and therefore has little effect on PII biosyn-thesis.

Here, we found that although addition of L-Phg to the �paaRmutant could partially rescue PI production, surprisingly, L-Phgsupplementation led to enhanced PII titers in the mutant (Fig. 6).Furthermore, interestingly, L-Phg addition to the parental strainled to a significantly enhanced PII production but reduced PIproduction. As we know, L-Phg formation probably initiates fromphenylpyruvate (PP) (Fig. 1) (5, 6), a compound from the shiki-mate pathway, which is the common pathway for the biosynthesisof aromatic compounds in bacteria and plants, such as L-trypto-

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phan, L-tyrosine, and L-phenylalanine. In bacteria, the activity ofkey enzymes of the shikimate pathway, including prephenate de-hydratase (PDT), chorismate mutase (CM), and 3-deoxy-D-ara-binoheptulosonate-7-phosphate (DAHP) synthase, is under strict

regulation via feedback inhibition. For instance, all three of thesearomatic amino acids could repress the activity of DAHP syn-thase, which is responsible for the formation of DAHP (the firstintermediate of the shikimate pathway) from erythose 4-phos-

FIG 7 Nucleotide sequences of the respective intergenic regions of paaH-paaZ, paaA-SSDG_03038, paaK-SSDG_07468, and bkdA-bkdR and their binding withpurified PaaR protein. (A) Nucleotide sequences of the respective intergenic regions of paaH-paaZ, paaA-SSDG_03038, paaK-SSDG_07468, and bkdA-bkdR. Theperfect conserved PaaR-binding motif is shaded, and the imperfect motif is indicated with broken lines. The transcriptional start sites (TSSs) are indicated by bentarrows. The putative �35 and �10 regions of the promoters of bkdA, paaZ, paaA, and paaK are underlined. The translational start and stop codons are markedby boxes. The primers used for amplification of EMSA probes are also underlined. (B) EMSA analysis. The concentrations of purified His6-PaaR protein (PaaR)used (nanomolar) in the assays are as indicated. We also performed competition assays with the addition of 200-fold specific (unlabeled each probe, indicated byS) and nonspecific (sperm DNA, indicated by N). The probe carrying the intergenic region of SSDG_03653-SSDG_05964 (containing the possible paaI promoterregion) was tested as a negative control. (C) EMSA analysis in the presence of PA-CoA, PAA, or acetyl-CoA. The same concentration (200 nM) of purifiedHis6-PaaR protein (PaaR) was used. PA-CoA, PAA, or acetyl-CoA was added at a concentration of 0, 10, 25, 50, 100, 200, or 400 �M. Free DNA probes andPaaR-probe DNA complexes are indicated by arrows.

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phate (an intermediate from the pentose phosphate pathway[PPP]) and phosphoenolpyruvate (an intermediate from the gly-colytic pathway) (19). This would reduce the metabolic flux to thebiosynthesis of aromatic amino acids; on the other hand, it wouldenhance the metabolic flux to the PPP pathway for a greaterNADPH supply as well as to the glycolytic pathway for the forma-tion of acetyl-CoA and then malonyl-CoA. If this is also the casefor S. pristinaespiralis, the addition of a high concentration of L-Phg (10 mg/25 ml) may result in a similar end product inhibition(feedback inhibition) and thereby lead to greater malonyl-CoAformation and, finally, enhanced PII biosynthesis. In addition, thebiosynthetic pathway of DMAPA, one of the PI amino acid pre-cursors, starts from chorismic acid, which is also an intermediatefrom the shikimate pathway (5). The end product repressionwould result in less carbon flow toward the DMAPA biosyntheticpathway. This may explain the findings that addition of L-Phg tothe parental strain resulted in reduced PI biosynthesis and that itsaddition to the �paaR mutant could lead to only partial restora-tion of PI titers.

ACKNOWLEDGMENTS

This study was funded by the National High Technology Research andDevelopment Program of China (2012AA022107), the National NaturalScience Foundation of China (31121001, 31370081, and 31430004), andthe National Basic Research Program of China (2012CB721103). We alsoacknowledge the support of the SA-SIBS scholarship program.

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